TB diagnostic based on antigens from M. tuberculosis

Abstract

The present invention is based on the identification and characterization of a number of novel M. tuberculosis derived proteins and protein fragments. The invention is directed to the polypeptides and immunologically active fragments thereof, the genes encoding them, immunological compositions such as diagnostic reagents containing the polypeptides.

1. A nucleic acid fragment in isolated form which
 1) comprises a nucleic acid sequence which encodes a polypeptide which
 a) comprises an amino acid sequence as shown in SEQ ID NO: 2, 
 b) comprises a subsequence of the amino acid sequence as shown in SEQ ID NO:2 which has a length of at least 6 amino acid residues, said subsequence being immunologically equivalent to the polypeptide defined in a) with respect to the ability of evoking a protective immune response against infections with mycobacteria belonging to the tuberculosis complex or with respect to the ability of eliciting a diagnostically significant immune response indicating previous or ongoing sensitization with antigens obtainable from mycobacteria belonging to the tuberculosis complex, or 
 c) comprises an amino acid sequence having a sequence identity with the amino acid sequence defined in a) or the subsequence defined in b) of at least 70% and at the same time being immunologically equivalent to the amino acid sequence defined in a) with respect to the ability of evoking a protective immune response against infections with mycobacteria belonging to the tuberculosis complex or with respect to the ability of eliciting a diagnostically significant immune response indicating previous or ongoing sensitization with antigens obtainable from mycobacteria belonging to the tuberculosis complex; or comprises a nucleic acid sequence complementary thereto. 
 
 
     
     
       2. A nucleic acid fragment according to  claim 1 , wherein said polypeptide comprises an epitope for a T-helper cell. 
     
     
       3. A nucleic acid fragment according to  claim 1 , wherein the polypeptide is free from any signal sequence. 
     
     
       4. A nucleic acid fragment according to  claim 1 , wherein said polypeptide
 1) induces a release of IFN-γ from primed memory T-lymphocytes withdrawn from a mouse within 2 weeks of primary infection or within 4 days after the mouse has been re-challenge infected with mycobacteria belonging to the tuberculosis complex, the induction performed by the addition of the polypeptide to a suspension comprising about 200.000 spleen cells per ml, the addition of the polypeptide resulting in a concentration of 1-4 μg polypeptide per ml suspension, the release of IFN-γ being assessable by determination of IFN-γ in supernatant harvested 2 days after the addition of the polypeptide to the suspension, and/or 
 2) induces a release of IFN-γ of at least 300 pg above background level from about 1000,000 human PBMC (peripheral blood mononuclear cells) per ml isolated from TB patients in the first phase of infection, or from healthy BCG vaccinated donors, or from healthy contacts to TB patients, the induction being performed by the addition of the polypeptide to a suspension comprising the about 1,000,000 PBMC per ml, the addition of the polypeptide resulting in a concentration of 1-4 μg polypeptide per ml suspension, the release of IFN-γ being assessable by determination of IFN-γ in supernatant harvested 2 days after the addition of the polypeptide to the suspension; and/or 
 3) induces an IFN-γ release from bovine PBMC obtained from animals previously sensitized with mycobacteria belonging to the tuberculosis complex, said release being at least two times the release observed from bovine PBMC obtained from animals not previously sensitized with mycobacteria belonging to the tuberculosis complex. 
 
     
     
       5. A nucleic acid fragment according to  claim 1 , wherein the sequence identity in c) is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98%. 
     
     
       6. A nucleic acid fragment comprising a nucleic acid sequence which encodes a polypeptide according to  claim 1 , wherein said polypeptide is a fusion polypeptide comprising at least one amino acid sequence or subsequence as defined in  claim 1  and at least one fusion partner. 
     
     
       7. A nucleic acid fragment comprising a nucleic acid sequence which encodes a polypeptide according to  claim 6  wherein the fusion partner is selected from the group consisting of:
 (a) a polypeptide fragment obtained from a virulent mycobacterium; and 
 (b) at least one immunogenic portion, of the polypeptides in (a). 
 
     
     
       8. A nucleic acid fragment according to  claim 1 , which is a DNA fragment. 
     
     
       9. A nucleic acid fragment according to  claim 1 , which has a length of at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70 or at least 80 nucleotides. 
     
     
       10. A nucleic acid fragment according to  claim 1 , which is more than 70% identical with a nucleic acid fragment which has a nucleotide sequence as defined in SEQ ID NO: 1. 
     
     
       11. A vaccine comprising a nucleic acid fragment according to  claim 1 , the vaccine effecting in vivo expression of antigen by an animal, including a human being, to whom the vaccine has been administered, the amount of expressed antigen being effective to confer substantially increased resistance to infections with mycobacteria of the tuberculosis complex in an animal, including a human being. 
     
     
       12. A vaccine for immunizing an animal, including a human being, against tuberculosis caused by mycobacteria belonging to the tuberculosis complex, comprising as the effective component a non-pathogenic microorganism, wherein at least one copy of a DNA fragment comprising a DNA sequence encoding a polypeptide as defined in  claim 1  has been incorporated into the genome of the microorganism in a manner allowing the microorganism to express and optionally secrete the polypeptide. 
     
     
       13. A vaccine according to  claim 12 , wherein the microorganism is a bacterium. 
     
     
       14. A vaccine according to  claim 13 , wherein the bacterium is selected from the group consisting of the genera  Mycobacterium, Salmonella, Pseudomonas  and  Escherichia.    
     
     
       15. A vaccine according to  claim 14 , wherein the microorganism is Mycobacterium Bovis BCG. 
     
     
       16. A vaccine according to  claim 15 , wherein at least 2 copies of a DNA fragment comprising a DNA sequence encoding a polypeptide have been incorporated into the genome of the microorganism, wherein the polypeptide
 a) comprises an amino acid sequence as shown in SEQ ID NO: 2, 
 b) comprises a subsequence of the amino acid sequence as shown in SEQ ID NO:2 which has a length of at least 6 amino acid residues, said subsequence being immunologically equivalent to the polypeptide defined in a) with respect to the ability of evoking a protective immune response against infections with mycobacteria belonging to the tuberculosis complex or with respect to the ability of eliciting a diagnostically significant immune response indicating previous or ongoing sensitization with antigens obtainable from mycobacteria belonging to the tuberculosis complex, or 
 c) comprises an amino acid sequence having a sequence identity with the amino acid sequence defined in a) or the subsequence defined in b) of at least 70% and at the same time being immunologically equivalent to the amino acid sequence defined in a) with respect to the ability of evoking a protective immune response against infections with mycobacteria belonging to the tuberculosis complex or with respect to the ability of eliciting a diagnostically significant immune response indicating previous or ongoing sensitization with antigens obtainable from mycobacteria belonging to the tuberculosis complex; or comprises a nucleic acid sequence complementary thereto. 
 
     
     
       17. A vaccine according to  claim 16 , wherein the number of copies is at least 5. 
     
     
       18. A replicable expression vector which comprises a nucleic acid fragment according  claim 1 . 
     
     
       19. A vector according to  claim 18 , which is an autonomously replicating vector. 
     
     
       20. A vector according to  claim 19 , which is selected from the group consisting of a virus, a bacteriophage, a plasmid, a cosmid and a microchromosome. 
     
     
       21. A vector according to  claim 18 , which is able to become integrated into the genome of the host cell. 
     
     
       22. A vector according to  claim 18 , which comprises a DNA segment encoding a reporter gene product useful for identification of heterologous gene products and/or a resistance gene such as an antibiotic resistance gene. 
     
     
       23. A transformed cell harboring at least one vector according to  claim 18 . 
     
     
       24. A transformed cell according to  claim 23 , which is a bacterium belonging to the tuberculosis complex, such as a  M. tuberculosis bovis  BCG cell. 
     
     
       25. A transformed cell according to  claim 23 , which expresses a polypeptide comprising:
 a) an amino acid sequence as shown in SEQ ID NO: 2, 
 b) a subsequence of the amino acid sequence as shown in SEQ ID NO:2 which has a length of at least 6 amino acid residues, said subsequence being immunologically equivalent to the polypeptide defined in a) with respect to the ability of evoking a protective immune response against infections with mycobacteria belonging to the tuberculosis complex or with respect to the ability of eliciting a diagnostically significant immune response indicating previous or ongoing sensitization with antigens obtainable from mycobacteria belonging to the tuberculosis complex, or 
 c) an amino acid sequence having a sequence identity with the amino acid sequence defined in a) or the subsequence defined in b) of at least 70% and at the same time being immunologically equivalent to the amino acid sequence defined in a) with respect to the ability of evoking a protective immune response against infections with mycobacteria belonging to the tuberculosis complex or with respect to the ability of eliciting a diagnostically significant immune response indicating previous or ongoing sensitization with antigens obtainable from mycobacteria belonging to the tuberculosis complex; or comprises a nucleic acid sequence complementary thereto. 
 
     
     
       26. A method for producing a polypeptide as defined in  claim 1 , comprising inserting a nucleic acid fragment according to  claim 1  into a vector which is able to replicate in a host cell, introducing the resulting recombinant vector into the host cell, culturing the host cell in a culture medium under conditions sufficient to effect expression of the polypeptide, and recovering the polypeptide from the host cell or culture medium; or isolating the polypeptide from a short-term culture filtrate as defined in  claim 1 ; or isolating the polypeptide from whole mycobacteria of the tuberculosis complex or from lysates or fractions thereof; or synthesizing the polypeptide by solid or liquid phase peptide synthesis. 
     
     
       27. A method for immunizing an animal, including a human being, against tuberculosis caused by mycobacteria belonging to the tuberculosis complex, comprising administering to the animal the vaccine according to  claim 13 . 
     
     
       28. A method according to  claim 27 , wherein the vaccine is administered by the parenteral (such as intravenous and intraarterially), intraperitoneal, intramuscular, subcutaneous, intradermal, oral, buccal, sublingual, nasal, rectal or transdermal route. 
     
     
       29. A composition for diagnosing tuberculosis in an animal, including a human being, comprising a nucleic acid fragment according to  claim 1 , optionally in combination with a means for detection. 
     
     
       30. A method for determining the presence of mycobacterial nucleic acids in a sample, comprising incubating the sample with a nucleic acid fragment according to  claim 1 , and detecting the presence of hybridized nucleic acids resulting from the incubation. 
     
     
       31. A method for effecting in vivo expression of an antigen by an animal comprising administering thereto the nucleic acid sequence according to  claim 1 , where in said expression confers resistance to infections with mycobacteria of the tuberculosis complex. 
     
     
       32. The fusion partner of  claim 7 , wherein said polypeptide fragment is selected from the group consisting of ESAT-6, MPB64, MPT64, TB10.4, CFP10, RD1-ORF5, RD1, ORF2, Rv1036, Ag85A, Ag85B, Ag85C, 19 KDa lipoprotein, MPT32, MPB59 and alpha-crystallin. 
     
     
       33. A nucleic acid fragment comprising a nucleic acid sequence which encodes a polypeptide according to  claim 6  wherein the fusion partner is selected from the group consisting of:
 a) a polypeptide, which comprises an amino acid sequence as shown in SEQ ID NO: 2, 
 b) a polypeptide, which comprises a subsequence of the amino acid sequence as shown in SEQ ID NO:2 which has a length of at least 6 amino acid residues, said subsequence being immunologically equivalent to the polypeptide defined in a) with respect to the ability of evoking a protective immune response against infections with mycobacteria belonging to the tuberculosis complex or with respect to the ability of eliciting a diagnostically significant immune response indicating previous or ongoing sensitization with antigens obtainable from mycobacteria belonging to the tuberculosis complex, or 
 c) a polypeptide, which comprises an amino acid sequence having a sequence identity with the amino acid sequence defined in a) or the subsequence defined in b) of at least 70% and at the same time being immunologically equivalent to the amino acid sequence defined in a) with respect to the ability of evoking a protective immune response against infections with mycobacteria belonging to the tuberculosis complex or with respect to the ability of eliciting a diagnostically significant immune response indicating previous or ongoing sensitization with antigens obtainable from mycobacteria belonging to the tuberculosis complex; or comprises a nucleic acid sequence complementary thereto. 
 
     
     
       34. A vaccine according to  claim 15 , wherein the microorganism is  Mycobacterium bovis  BCG strain: Danish 1331. 
     
     
       35. A method for producing a polypeptide as defined in  claim 1 , comprising inserting a nucleic acid fragment according to  claim 1  into a vector which is able to replicate in a host cell, introducing the resulting recombinant vector into the host cell, culturing the host cell in a culture medium under conditions sufficient to effect expression of the polypeptide, and recovering the polypeptide from the host cell or culture medium; or isolating the polypeptide from a short-term culture filtrate as defined in  claim 1 ; or isolating the polypeptide from cell wall containing fractions of the tuberculosis complex. 
     
     
       36. A nucleic acid fragment according to  claim 1 , wherein the sequence identity in c) is 100%. 
     
     
       37. A nucleic acid fragment according to  claim 7 , wherein the immunogenic portion is a T-cell epitope.